The efficiency of a nucleic acid modifying enzyme, i.e., the amount of modified product generated by the enzyme per binding event, can be enhanced by increasing the stability of the modifying enzyme/nucleic acid complex. The prior art has suggested that attachment of a high probability binding site, e.g., a positively charged binding tail, to a nucleic acid modifying enzyme can increase the frequency with which the modifying enzyme interacts with the nucleic acid (see, e.g., U.S. Pat. No. 5, 474,911). The present invention now provides novel modifying enzymes in which the double-stranded conformation of the nucleic acid is stabilized and the efficiency of the enzyme increased by joining a sequence-non-specific double-stranded nucleic acid binding domain to the enzyme, or its catalytic domain. The modifying proteins that are processive in nature exhibit increased processivity when joined to a binding domain compared to the enzyme alone. Moreover, both processive and non-processive modifying enzymes exhibit increased efficiency at higher temperatures when joined to a typical binding domain described herein.
In one aspect, the invention provides fusion polymerases that provide an enhanced ability to perform long PCR, i.e., amplification of sequences of 5 kb or greater in length. Polymerase mixture to perform long PCR are known in the art. One difficulty with such mixtures is that long extension times are required for optimal yields of the expected products. Conventional recommendations are to allow extension times of 1 minute per kb. For a 10 kb product and using 40 cycles of amplification, the PCR requires approximately seven hours. Described herein are modified polymerase enzymes that exhibit an enhanced ability to perform long PCR compared to those polymerases and/or polymerase mixtures currently available. These modified enzymes, e.g., Pfu-Sso7d, incorporate a polymerase with error-correcting activity and a sequence-nonspecific double-stranded DNA-binding domain, and therefore provide the capability of PCR amplifying DNA larger than 5 kb without need to resort to polymerase mixtures. In addition, such modified enzymes can efficiently amplify a given fragment using shorter extension times than are required by conventional polymerase mixtures. Thus, use of the hybrid polymerases described herein instead of the conventional mixtures can greatly decrease the reaction time required to amplify large fragments, e.g. from about seven hours to about two hours.